The breast oncogene ErbB-2 is amplified or overexpressed in 15-30% of breast cancers. ErbB-2 overexpression is associated with resistance to cytotoxic and anti-hormone treatment and poor overall survival. Anti-ErbB-2 treatment strategies include the use of trastuzumab, a humanized monoclonal antibody directed against ErbB-2, plus a taxane-based chemotherapy or lapatinib, a dual EGFR and ErbB-2 tyrosine kinase inhibitor-approved for trastuzumab resistant disease. Unfortunately, resistance to trastuzumab and now lapatinib is a major problem in metastatic breast cancer with only 30-50% responding to anti-ErbB-2 therapy. Therefore, the identification of compensatory survival pathways that contribute to anti-ErbB-2 treatment resistance is important in order to design rational targeted treatment options to prevent or reverse resistance and improve overall survival. We have identified that Notch-1, another potent breast oncogene and cell fate determinant, is activated by trastuzumab or a TKI similar to lapatinib in ErbB-2 positive breast cancer cells and served as a novel therapeutic target for anti-ErbB-2 treatment resistance (1). Our findings suggested that there is a compensatory increase in Notch-1-mediated proliferation/survival in trastuzumab or a TKI similar to lapatinib-treated breast cancer (1). The Notch-1 receptor is another potent breast oncogene (2) that is overexpressed with its ligand Jagged-1 in breast cancers associated with the poorest overall survival (3-5). Furthermore, Notch has been shown to be necessary for the proliferation and survival of breast cancer stem cells (6). Therefore, it i critical to identify the mechanism(s) by which ErbB-2 inhibition activates Notch-1 activity and whether suppression of the Notch pathway prevents and/or reverses resistance to trastuzumab or lapatinib in vivo in order to develop the optimal therapy for women with ErbB-2 positive breast cancer. Based on our findings, we propose a central hypothesis that ErbB-2 overexpression and activity de-regulates Jagged- 1 vesicular transport restricting Jagged-1-mediated Notch activation at the cell surface. Thus, resistance to anti-ErbB-2-targeted agents can be prevented and/or reversed by targeting Jagged-1 and/or Notch-1 directly. We have formulated this hypothesis based on our preliminary data suggesting that hyperactive ErbB-2 de- regulates vesicular trafficking of Jagged-1, and that Jagged-1 was required for trastuzumab-induced increase in Notch-1 transcriptional activity. To test our hypothesis and achieve the overall objective, we propose three Specific Aims: Specific Aim 1: Identify the molecular mechanism(s) responsible for Jagged-1-mediated Notch-1 activation in response to ErbB-2 inhibition. Our working hypothesis based on strong preliminary data is, that hyperactive ErbB-2 restricts proper Jagged-1 endocytosis to inhibit Notch activation at the cell surface. ErbB-2 inhibition rescues this effect, resulting in re-activation of Jagged-1-mediated Notch signaling. Specific Aim 2: Determine if Jagged-1 is the critical mediator of Notch Signalling to confer resistance to anti-ErbB-2 treatment in vitro. Our working hypothesis is that availability of cell surface Jagged-1 is critical to activating Notch-1 and thus promoting cell proliferation, survival, and resistance to anti-ErbB-2 treatment in vitro. Specific Aim 3: Evaluate the therapeutic efficacy of targeting Jagged-1 to prevent tumor recurrence and reverse resistance in vivo. Our working hypothesis is that simultaneous targeting of both ErbB-2 and Jagged-1-mediated Notch signaling pathways will prevent and/or reverse trastuzumab or lapatinib resistant ErbB-2 positive breast cancer in vivo providing pre-clinical evidence for a future prospective efficacy trial. Upon successful completion of these studies, we will begin a clinical trial to test our pre-clinical results. Our gal is development of scientifically-based evidence to support a therapeutic benefit of combining anti- ErbB-2 treatment with Notch pathway inhibition which provides a foundation for Phase I/Phase 2 clinical trials to test benefits in women with ErbB-2 positive breast cancer with the goal of preventing or reversing resistance.